Figure 2: Evaluation of matrix effects. Six negative patient samples (3 male and 3 female) were prepared without adding the IS. Upon injection, a constant flow (5 μL/min) of each analyte (1 μg/mL prepared in methanol) was infused post-column into the mobile phase using a T junction. MRMs of each analyte were monitored for the entire LC gradient. A representative chromatogram of a patient (solid line) and a blank injection of water (dashed line) monitored for the primary transition are shown. Retention time of each analyte is indicated with an arrow.